The clearance of drugs, toxins, environmental contaminants and other waste products from the body often involves processing in the liver to form glucuronide conjugates, which are more readily solubilized and excreted by the kidneys. Any studies monitoring the processing of these metabolites must either measure both free and conjugated forms of the analytes or the conjugates must be hydrolyzed to allow determination of total excreted analytes in the urine.
This report describes a completely automated “Prep-and-Shoot” workflow in a 96 well plate format for the analysis of multiple drug classes in urine samples. A GERSTEL MultiPurpose Sampler (MPS) coupled to an AB SCIEX QTRAP® 4500 LC/MS/MS system was used for a fast enzymatic hydrolysis process, dilution, and injection of urine samples. Over 40 drugs and their metabolites were monitored using the Scheduled MRM™ algorithm programmed in the LC/MS/MS acquisition method.
Results and Discussion
The efficiency of the automated enzymatic hydrolysis was examined by spiking blank urine with 8 different glucuronide conjugates. The use of the β-Glucuronidase enzyme in combination with the optimized rapid buffering solution ensured completion of the hydrolysis procedure within 15 minutes.
Over 95 % hydrolysis was achieved for most compounds with the exception of Codeine-6-Glucuronide (80.5 % hydrolyzed), which required a longer incubation time for complete deconjugation.
The robustness of the analytical column was tested by injecting approximately 960 hydrolyzed and diluted urine samples and by measuring the column back pressure as a function of the number of injections made on the column. The Phenomenex Security guard column was replaced after the analysis of every 2 plates, resulting in an average back pressure of 1478.3 psi with a % RSD of 4.3 % indicating no adverse pressure buildup due to fouling on the analytical column.
A qualitative assessment of the mass spectrometer inlet was performed before and after the robustness testing. The simple source architecture and orthogonal spray design of the AB SCIEX Turbo V™ ion source provides outstanding robustness and sensitivity for complex biological matrices. The AB SCIEX Turbo V™ ion source, therefore, in combination with the ultra-clean hydrolysis enzyme, allows for this simple, high throughput “Prep-and-shoot” approach.
Ten incurred urine samples with semi quantitative results were automatically hydrolyzed and injected into the LC/MS/MS system. Each sample was hydrolyzed, diluted and injected a total of 96 times. In each chromatogram the glucuronide conjugate and parent drug ions monitored were extracted to show the completion of the 15 minute enzymatic hydrolysis process.
As a result of this study, we were able to show:
• A fast and simple “Prep-and-Shoot” workflow was developed using the GERSTEL MPS autosampler and sample preparation robot coupled to an AB SCIEX QTRAP® 4500 LC/MS/MS System for the automated hydrolysis and analysis of urine samples within a single automated run.
• The buffer, internal standard and β-Glucuronidase combination was optimized to perform the automated urine hydrolysis, yielding hydrolysis efficiencies above 80 % with a 15 minute incubation time.
• The combination of LC/MS/MS analysis conditions, the ultra-high purity of the β-Glucuronidase and the design of the Turbo V™ ionization source, allowed over 960 urine samples to be automatically hydrolyzed, diluted injected, and analyzed without having to replace the analytical column while delivering reproducible results.
• The combined automation of urine hydrolysis, injection and analysis using the GERSTEL MPS allowed the system to process more than 200 samples in a 24 hour period.